The biotechnology industry has progressed over the years from relatively simple lab benches to highly complex manufacturing processes. The industry has adopted manufacturing techniques and equipment from various existing processes and modified such processes to meet certain distinct uses. For example, in-line optical monitoring instrumentation (originally developed for applications ranging from brewing to chemical process) has been modified for use in the biotechnology field.
The biotechnology industry has evolved considerably with the development of new processing equipment and techniques to improve yields, ensure regulatory compliance, and increase production capabilities. However, optical monitoring instrumentation, which is an integral part of nearly every production process in the biotechnology industry, has not kept pace with this trend. Current in-line optical instrumentation is functionally no different than the instrumentation adopted from other industries many years ago.
Most modern biotechnology manufacturing facilities use three distinct inline optical monitoring technologies to help monitor and control their processes. Near infrared (NIR) absorption sensors are used primarily to monitor cell density in bioreactors and to measure certain concentrations in various separation stages, such as centrifugation, perfusion, and filtration. Scattered light turbidmeters are used to detect low concentrations solids carryover in the purified centrate or filtrate streams from these separation processes. Further downstream, ultraviolet (UV) absorption sensors are used to measure protein concentrations on the outlet of chromatography columns.
The protein concentration measurement using UV absorption sensors is one of the primary control parameters in the entire manufacturing process. A UV absorption sensor typically comprises an inline flow cell with quartz or sapphire windows and lamp and detector assemblies mounted on opposing sides of the flow cell. The distance between the windows provides the optical path length (OPL). The protein concentration can be measured or otherwise determined by directing light through the windows, and then determining the amount of light absorbed by the material (e.g., protein) that is present between the windows.
A variable that is available to adjust the range of measurement is the OPL. Increasing the OPL improves sensitivity but reduces the maximum measurement range. Decreasing the OPL has the inverse effect. This need for changes in the OPL can be driven by a process change that results in higher protein concentrations or the switch to an entirely different product, for instance, which happens quite frequently in process development and manufacturing facilities.
To achieve a desired OPL, current online UV sensors employ a combination of optical windows of different thickness with flow cells of varying internal dimensions, thereby obtaining a range of discrete OPLs available for a given line size. Typically, available OPLs range from 1-10 mm in 1-2.5 mm increments for a given UV sensor. Regardless of the particular OPL for a given UV sensor, the commonality is that the OPL is fixed for any given sensor.
One of the drawbacks of current UV sensors is that even minor OPL changes require time-consuming and sometimes expensive hardware changes. For instance, to change the OPL requires, at a minimum, replacement of the optical windows, and quite frequently, replacement of the flow cell as well. To replace both optical windows and the flow cell can cost $2000-$2500. More importantly, if the specific required flow cells are unavailable from a vendor or other supplier, this switch can delay processing for 4-6 weeks or other significant amount of time.
As an illustration with the currently available UV sensors, a combination of three different flow cells and up to six different windows might be required to achieve all available optical path lengths from 1-10 mm for a given line size. In order to maintain maximum flexibility, a process development facility, vendor, or other entity might have to stock all of these parts. Furthermore, if a manufacturing facility needs to inventory all possible spares (which is typical), the manufacturing facility would usually have to stock numerous flow cells and numerous window combinations to cover all of their applications. Maintaining this inventory also has additional associated costs based on the requirement of traceability of materials. This procedure is not just a matter of grabbing a flow cell off of a shelf, but rather also involves locating and recording all appropriate paperwork and certifications.
Existing UV sensors also have significant drawbacks in precision/accuracy. With traditional hardware, the accuracy of the OPL is determined by three parameters: the distance between the window seats on the flow cell as well as the thickness of each of the two optical windows. Using tightly controlled manufacturing methods, current manufacturers have been able to maintain the tolerances on each of these dimensions to ±0.05 mm. This means that a cumulative error of ±0.15 mm is still within tolerance. Probably the most common optical path length for chromatography applications is 1 mm, which means that the stated tolerances could result in an error of ±15%. The worst-case scenario would have the readings on redundant UV sensors deviating from one another by 30% (one +15%, one −15%), and still being within tolerance. Typically, when manufacturers use redundant sensors, the sensor readings have to be within a certain tolerance of one another (usually 5-10%) or the run will have to be terminated. Because of the OPL deviations, several manufacturers have had to modify their sensor readings with a “fudge factor” to keep the readings within tolerance. These deviations are impractical in situations where a precise determination of OPL is desirable.
On the UV sensors currently used in the industry, there is no external indication of optical path length. This makes it difficult, if not impossible, to determine the actual OPL of an installed UV sensor without removing the flow cell from the process, and in many cases, completely disassembling it. This problem has led to many instances of faulty calibration based on incorrect assumptions about optical path length. Numerous situations exist where users have assumed that they are operating with a certain OPL (for instance 2 mm), when in reality, the manufacturer has provided a UV sensor with a different OPL (for instance 1 mm OPL). Also, because the windows for different optical path length are identical in appearance except for small variations in length (sometimes as small as 0.5 mm, for instance), it is extremely easy for users to mismatch windows when re-assembling flow cells after cleaning or service, again resulting in incorrect OPLs. Since there is no external indication of optical path length, this type of problem can take many hours to resolve. Because many of the line sizes used are too small to allow use of a ball mill or other mechanism to directly measure the OPL, the only way to reliably determine OPL is to completely disassemble the flow cell, measure the window thicknesses with calipers, and then consult with the factory to determine what OPL that window combination and flow cell will yield. Again, these hours are often inside of a controlled clean room. In the worst-case scenario, the manufacturer actually makes product using the improperly configured sensor to control cuts.
Other costs are associated with current UV sensors having fixed OPLs. For instance, there is also the additional cost of bringing the UV sensor back into a clean environment and re-sterilizing after an OPL change has been made. Clearly therefore, current UV sensors having fixed OPLs are impractical in many situations.